PROJECT SUMMARY Alternative splicing is a key mechanism for regulating genetic output that is directed by diverse pre-mRNA binding proteins. Although recent genomic analyses have lent insight into the breadth of the regulatory networks controlled by these proteins, our mechanistic understanding of the process is rudimentary. Little is known of the molecular interactions by which regulatory proteins affect the assembling spliceosome, and such information is essential to understanding the many forms of human disease attributed to misregulated splicing. This project will study the Rbfox RNA binding proteins that control the splicing of many transcripts important for neuronal function and synaptic activity, and which are implicated in epileptic and autism spectrum disorders. We recently showed that the nuclear Rbfox isoforms are bound with a novel macromolecular complex containing eight other RNA binding proteins and called a large assembly of splicing regulators, LASR. Virtually all the Rbfox protein bound to unspliced RNA is associated with a LASR complex, and data indicate that Rbfox functions with LASR to control splicing. We now propose to characterize Rbfox/LASR interactions and activity in detail. Using in vivo and in vitro assays, we will identify protein-protein interactions necessary for LASR assembly from its subunits, for Rbfox association, and for its multimerization into higher order complexes. We will characterize the protected RNA sequences that copurify with LASR and will define which fragments associate with particular subunits. Genomewide iCLIP analysis will map the binding of LASR subunits relative to the known Rbfox binding sites. With the goal of understanding how it is targeted to particular RNA features, we will test the binding of Rbfox/LASR and purified LASR subunits to individual motifs and to combinations of motifs in vitro. Using splicing reporter genes and genomewide RNAseq assays, we will define the common targets of splicing regulation by LASR and Rbfox. Finally, we will examine how Rbfox and individual LASR subunits cooperate in regulating particular target exons using CRISPR knockout cell lines and RNAi in neurons. These studies will yield new understanding of the intricate combinations of RNA elements and binding proteins that mediate the regulation of splicing, and its misregulation in human disease.